Monitoring NAD+ levels in aging using a novel genetically-encoded biosensor

项目摘要 烟酰胺腺嘌呤二核苷酸(NAD+)是sirtuins和PolyADP-核糖聚合酶的底物 (PAPS)，将其与基因表达和基因组稳定性联系起来。这些酶活性也将NAD+连接到 如糖尿病和肌肉无力等与衰老相关的疾病。NAD+更改也是最重要的 卡路里限制(CR)和疾病预防之间的关系，但这种联系的确切性质仍然存在 未知。一种模型是，CR升高细胞内NAD+，然后控制sirtuins的活性，从而 调节燃料利用和营养反应基因的表达。PARP激活也会影响 通过消耗NAD+来维持细胞能量的动态平衡，最终导致细胞死亡。记住这一点很重要 NAD+对sirtuins和parps的贡献完全取决于游离NAD+浓度和 不是氧化还原，也就是NAD+/NADH比率。尽管测量NAD+/NADH比率很简单， 监控NAD+并非如此--我们开发的NAD+生物传感器首次让人们得以一窥NAD+ 在完整细胞的亚细胞室内的调节。这是一个重要的进步，因为之前 研究不能区分游离和绑定的NAD+，也不能监测不同国家之间NAD+调控的差异 车厢。有了这个新的传感器，我们将确定NAD+在胰腺β细胞和 骨骼肌在衰老过程中是否受到调节，以及与年龄相关的变化是否可以通过CR或 NAD+产量的增加。虽然我们已经使用NAD+生物学获得了重要的见解 电流传感器的出现，对于将我们的研究扩展到完整的动物身上将具有重要的价值。因此，我们的第一个目标是 建立表达NAD+生物传感器的条件转基因小鼠系。我们已经取得了重大进展 优化我们的传感器以进行活体动态测量的进展，并描述了提高其 灵敏度和动态范围进一步提高。然后我们将测试老化是否减少，CR或NAD+前体 给药增加了胰岛β细胞和骨骼肌细胞中NAD+的水平，这是通过产生组织- 能够监测细胞核、细胞质和线粒体中NAD+水平的特定传感器菌株。NAD+ 耗竭被认为是与年龄相关的胰岛素分泌减少的中介。同样，与年龄相关的NAD+ 减少被认为是肌肉无力和肌肉再生能力受损的基础。 我们的生物传感器提供了一个前所未有的机会来检测衰老对NAD+水平的影响， NAD+对年龄相关疾病的贡献以及几种建议的改善方法的有效性 这些条件。

Project Summary Nicotinamide adenine dinucleotide (NAD+) is the substrate for sirtuins and polyADP-ribose polymerases (PARPs), linking it to gene expression and genomic stability. These enzymatic activities also connect NAD+ to such aging-related conditions as diabetes and muscle weakness. NAD+ alterations also figure prominently in the relationship between calorie restriction (CR) and disease prevention, but the exact nature of this link remains unknown. One model is that CR elevates intracellular NAD+, which then controls activity of sirtuins that regulate fuel utilization and expression of nutrient-responsive genes. PARP activation can also influence cellular energy homeostasis by depleting NAD+, ultimately leading to cell death. It is important to remember that the contribution of NAD+ to sirtuins and PARPs depends entirely upon the free NAD+ concentration and not on redox, that is, the NAD+/NADH ratio. Although measuring the NAD+/NADH ratio is straightforward, monitoring NAD+ is not—our development of an NAD+ biosensor has provided the first glimpses into NAD+ regulation within subcellular compartments of intact cells. This is an important advance because previous studies could not distinguish free from bound NAD+ or monitor differences in NAD+ regulation across compartments. With this novel sensor in hand, we will determine how NAD+ levels in pancreatic beta cells and skeletal muscle are regulated during aging and whether age-related changes can be prevented by CR or augmentation of NAD+ production. Although we have gained significant insights into NAD+ biology using the current sensor, it would be of great value to extend our studies into intact animals. Thus, our first goal is to develop a conditional transgenic mouse line expressing the NAD+ biosensor. We have already made significant progress optimizing our sensor for in vivo dynamic measurements and describe strategies for increasing its sensitivity and dynamic range further. We will then test whether aging decreases, and CR or NAD+ precursor administration increases, NAD+ levels in pancreatic beta cells and skeletal muscle cells by generating tissue- specific sensor strains capable of monitoring NAD+ levels in the nucleus, cytoplasm, and mitochondria. NAD+ depletion is thought to mediate age-related decreases in insulin secretion. Similarly, age-dependent NAD+ decreases have been proposed to underlie muscle weakness and impairments in muscle regenerative capacity. Our biosensor provides an unprecedented opportunity to examine the effect of aging on NAD+ levels, the contribution of NAD+ to age-related disorders, and the efficacy of several proposed approaches to ameliorating these conditions.